1. Field of the Invention
This invention relates to a novel glycolipid derivatives thereof which have effective anti-tumor activity and immunostimulating activity thereof and the production method thereof, further relates to a medicine composition containing the glycolipid derivatives thereof.
2. Description of the Prior Art
A glycolipid play important biological roles to cell deffarantation, proliferation, and navus cell growth. (Hakomori et al, Annu. Rev. Biochem. 1981, 50, 733-764.; Morita et al, Glycosphingolipids. 1985, 59-82, Elsevier Science Publishing Co., New York.). And, Morita et al (J. Med. Chem. 1991, 38, 2176.) and Kawano et al (Proc. Natl. Acad. Sci. USA. 1998, 95, 5690.) have been reported that alpha-galactosylceramides have effective anti-tumor activity and immunostimulating activity. Futhermore, Kawano et al (Science. 1997, 278, 1626-1629.) and Sakai et al (J. Med. Chem. 1999, 42, 1836-1841.) have been reported that alpha-galactosylceramides exhibit stronger antitumor activity and immunostimulating activity than beta-galactosylceramide.
However, when the galactosylceramide is administrated as a medicine, we expect that glycosyl bond is hydrolyzed by enzyme so as galactosidase which exist in a lysozome (Chen et al., Biochem. Biophys. Acta. 1993, 1170, 53-61). C-glycosides, that oxygen atom on a glycoside bond were exchanged to carbon atom, has been known as a stable sugar derivatives on a glycosidase metabolism (Linhardt et al., Tetrahedron. 1998, 54, 9913-9959; Levy et al., The Chemistry of C-Glycosides, Pergamon:Oxford, 1995; Postema et al, C-Glycoside Synthesis, CRC Press: Boca Raton, 1995). In addition, S-glycoside which exchange oxygen atom to sulfur atom on the glycoside bond (Rahman et al, In Studies in Natural Products Chemistry, Elsevier: New York, 1991; Vol. 8, 315-317; Defaye et al, J. Carbohydr. Chem. (UK) 1998, 30, 159-166.) and N-glycosides which oxygen atom were replaced by nitrogen atom (Stunkel et al., Prog. Leukocyte Biol. 1989, 9, 575-579.) have been also known.
As a synthesis of the C-glycosides [which] with oxygen on the glycoside bond of a galactosyl ceramide were replaced by carbon atom, synthesis of the beta-C-galactosyl ceramide has been only report (Dondoni et al., J. Org. Chem. 1991, 64, 5557-5564). However, synthesis of a alpha-C-galactosyl ceramide (alpha-compound) has not been reported. Owing to stabilities against alpha-galactosidase, the alpha-compounds which have alpha-C, N, or S-glycoside bond are expected more effective than the alpha-O-galactoside that is decomposed by a alpha-galactosidase. Thus, those compounds have a potent anti-cancer activity and an immunostimulating activity, furthermore, are stable against hydrolysis with glucosidase, acid, and base, and also able to keep at a room temperature for a long time.
3. Problems to be Solved by the Invention
The object of the present invention is the provision of the glycolipid derivatives exhibiting potent antitumor activity and immunostimulating activity, and having alpha-C, N, or S-glycoside bond for allowing to stand at room temperature for a long time, Thus, the glycolipid derivatives exhibit potent antitumor activity and immunostimulating activity than a known compound, and furthermore it is able to save at room temperature for long time and continue the activity for long time.
4. Methods for Solution of the Problems
As a result of that research a glyoclipid derivatives for the purpose of synthesis of anti-tumor agent and immunostimulating agent, we found that a glycolipid derivatives which is described in the general formula (I), exhibits a potent anti-tumor effect and immunostimulating effect.
The present invention provides medical composition which containing the general formula (I) and its pharmaceutically acceptable salt as active ingredient. 
wherein W represents carbon chain from 9 to 17 which containing double bond or hydroxy group occasionally; X represents carbon chain from 11 to 25 which containing double bond or hydroxy group occasionally; Y represents xe2x80x94(CH2)axe2x80x94CHxe2x95x90CHxe2x80x94(CH2)axe2x80x94, xe2x80x94(CH2)axe2x80x94(a,axe2x80x2 denotes an integer of 0-5 and a+axe2x80x2 is 5 and under [.]), xe2x80x94S(O)0-2CH2xe2x80x94, xe2x80x94NHCH2xe2x80x94,; Z represents xe2x80x94COxe2x80x94, xe2x80x94SO2xe2x80x94; R represents xe2x80x94CH2OH, xe2x80x94CO2H, xe2x80x94CH2OCH2CO2H, xe2x80x94CH2OSO3H; R0 represents xe2x80x94OH, xe2x80x94NH2, xe2x80x94NHAc.
A preparing method of general formula (I) in the present invention are examplified and not restrict the disclosed invention. And, each sign are as mentioned above.
(A) Synthesis of the Ceramide Moiety
I. The ceramide precursor (A-1) is prepared by Schmidt""s method (Liebigs Ann. 1995, 755-764) using D-galactose as a starting material. A primary hydroxy group of the compound (A-1) is tritylated, followed by benzylation of the secondary hydroxy group, and the conpound (A-2) is obtianed. 
(wherein W1 is a carbon chain of C8 to C16 and it contains saturated position as the case may be.)
An azide group of the compound (A-2) is reduced by hydrogenatoin, or triphenylphosphine and water, thus the amino compound (A-3) is obtained. 
The generated amino group is carried out amidation or sulfonylation in the presence of base, and the compound (A-4) or (A-5) are obtained, respectively. Furthermore, the compound (A-3) is condenced by coupling reagents (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.HCl salt (WSCDI) et cetera), and the compound (A-4) is also able to obtain. 
The compound (A-4) or (A-5) are reacted with acid for detritylation, to yield the compound (A-6) or (A-7). Then the generated hydroxy group is oxidized (Swern oxidation (J. Org. Chem. 1978, 43, 2482) etc), to yield the aldehyde compound (A-8) or (A-9). 
II. The compound (A-2) is reacted with acid for detritylation, followed by a reduction (PPh3/H2O etc) of an azide group, to yield the compound (A-10). Then amino group of the compound (A-10) is protected with Boc group, followed by an oxidation of the primary hydroxy group (Swern oxidation (J. Org. Chem. 1978, 43, 2482) etc), to yield the aldehyde compound (A-11). 
III. A primary hydroxy group of the compound (A-12) is carried out methanesulfonylation or bromination, to yield the compound (A-13). 
(B) Synthesis of the Sugar Moiety
A cyano group of galactosylcyano compound (B-1) is reduced by Lopex""s procedure (J. Carbohydr. Chem. 1987, 6, 273-279), to yield the alcohol compound (B-2). 
A primary hydroxy group of the compound (B-xe2x80x942) is carried out tosylation followed by iodation, to yield the compound (B-3). Then it is reacted with triphenylphosphine, to yield the compound (B-4).
The compound (B-4) is reacted with base (n-butyl lithium, potassium fluoride, etc), to yield the compound (B-5). 
(C) Coupling Reaction with the Sugar Moiety and the Ceramide Moiety
I. The ceramide compound (A-8) and (A-9) which was prepared by the method (A), is carried out Wittig reaction with the compound (B-5) which was prepared by the method (B), to yield the compound (C-1) or (C-2) Then, those are reacted with sodium metal in liquid ammonia to yield the compound (C-3) or (C-4) Furthermore, those are hydrogenated to yield the compound (C-5) or (C-6). As another method, the compound (C-1) and (C-2) are directly hydrogenated to yield the compound (C-5) or (C-6). 
II. The ceramide compound (A-11) which was prepared by the method (A), is carried out wittig reaction with the compound (B-5) which was prepared by the method (B), to yield the compound (C-7). Then, it is reacted with a sodium metal in liquid ammonia to yield the compound (C-8). Furthermore, it is hydrogenated to yield the compound (C-9). As another method, the compound (C-7) is directly hydrogenated to yield the compound (C-9). 
The compound (C-8) or (C-9) is reacted with acid for deprotection to yield the compound (C-10) or (C-11), then the amino group is carried out amidation or sulfonylation to yield the compound (C-3, (C-4), (C-5), or (C-6). 
III. The ceramide compound (A-13) which was prepared by the method (A), is glycosylated with the sugar derivatives (C-12) or (C-13) in the presence of base so as cesium carbonate, to yield the compound (C-14) or (C-15). 
The compound (C-14) or (C-15) are deprotected to yield the compound (C-16) or (C-17), then the amino group is carried out amidation or sulfonylation to yield the compound (C-18), (C-19), (C-20), or (C-21). Case by case, the compound (C-18) or (C-19) are oxidazed to yield the compound (C-22) or (C-23), respectively. 
(D) Modification of Primary Hydroxy Group of the Suger Moiety 
II. In the compound of general formula (I) when R is CH2OCH2CO2H, the compound (D-1) is reacted with BrCH2CO2H in the presence of NaOH to yield the compound (D-3). 
III. In the compound of general formula (I) when R is CO2H, a primary hydroxy group of the compound (D-1) is converted to carboxylic acid group with oxidant (TEMPO=2,2,6,6-tetramethyl-1-piperidinyloxy freeradical, etc) to yield the compound (D-4). 
The compounds related to the general formula (I) are exemplified as follows and not restrict the disclosed invention. And, the chemical structure and the analytical value are exhibited as following table.
(1). (3xe2x80x2S,4xe2x80x2S,5xe2x80x2R)-3xe2x80x2-N-Hexacosanoylamino-4xe2x80x2,5xe2x80x2-dihydroxy-nonadecane-xcex1-C-D-galactopyranoside (Compound 1)
(2). (3xe2x80x2S,4xe2x80x2S,5xe2x80x2R)-3xe2x80x2-N-Hexacosanoylamino-4xe2x80x2,5xe2x80x2-dihydroxy-1xe2x80x2-(E/Z)-nonadecane-xcex1-C-D-galactopyranoside (Compound 2)
(3). (3xe2x80x2S,4xe2x80x2S,5xe2x80x2R)-3xe2x80x2-N-Pentacosanesulfonylamino-4xe2x80x2,5xe2x80x2-dihydroxy-nonadecane-xcex1-C-D-galactopyranoside (Compound 3)
(4). (5xe2x80x2S,6xe2x80x2S,7xe2x80x2R)-6xe2x80x2,7xe2x80x2-Dihydroxy-5xe2x80x2-N-hexacosanoylamino-(E/Z)-henicos-3xe2x80x2-enyl-xcex1-C-D-galactopyranoside (Compound 4)
(5). (5xe2x80x2S,6xe2x80x2S,7xe2x80x2R)-6xe2x80x2,7xe2x80x2-Dihydroxy-5xe2x80x2-N-tetracosanoylamino-(E/Z)-henicos-3xe2x80x2-enyl-xcex1-C-D-galactopyranoside (Compound 5)
(6). (5xe2x80x2S,6xe2x80x2S,7xe2x80x2R)-6xe2x80x2,7xe2x80x2-Dihydroxy-5xe2x80x2-N-hexacosanoylamino-henicosane-xcex1-C-D-galactopyranoside (Compound 6)
(7). (5xe2x80x2S,6xe2x80x2S,7xe2x80x2R)-6xe2x80x2,7xe2x80x2-Dihydroxy-5xe2x80x2-N-tetracosanoylamino-henicosane-xcex1-C-D-galactopyranoside (Compound 7)
(8). (2xe2x80x2S,3xe2x80x2S,4xe2x80x2R)-3xe2x80x2,4xe2x80x2-Dihydroxy-2xe2x80x2-N-hexacosanoylamino-octadecane-1xe2x80x2-thio-yl-xcex1-C-D-galactopyranoside (Compound 8)
(9). (3xe2x80x2S,4xe2x80x2S,5xe2x80x2R)-3xe2x80x2-N-Hexacosanoylamino-4xe2x80x2,5xe2x80x2-dihydroxy-nonadecane-xcex1-C-D-(6-O-methylenecarboxylic acid)-galactopyranoside (Compound 9)
(10). (3xe2x80x2S,4xe2x80x2S,5xe2x80x2R)-3xe2x80x2-N-Hexacosanesulfonylamino-4xe2x80x2,5xe2x80x2-dihydroxy-nonadecane-xcex1-C-D-(6-O-methylenecarboxylic acid)-galactopyranoside (Compound 10)
(11). (3xe2x80x2S,4xe2x80x2S,5xe2x80x2R)-3xe2x80x2-N-Hexacosanesulfonylamino-4xe2x80x2,5xe2x80x2-dihydroxy-nonadecane-xcex1-C-D-galactulonic acid (Compound 11)
The compounds which are described general formula (I) in the present invention or the pharmaceutically acceptable salts can be administered to human or mammal. And the compounds can be formed injections, powders, granules, tablets, capsules, troches, dry-syrups, liposome prepapations etc by known technique to preparation or it""s own. The appropriate dose and dosage times, that of the compound pf the present invention, must be determined by the conditions of a patient, age, body weight etc.
A experimental example of anti-tumor activity and immunostimulating activity is described as follows:
Experiment 1: Lymphocyte mixed culture reaction.
The pancreatic cells of BALB/C mouse were regulated to a concentration of 2xc3x97106 cells/ml with a culture medium of 10% FCS RPMI 1640, respectively. These cells (100 ul/well) and a sample (10 ul/well) were plated in a 96 well round-bottomed plate and cultured for 3 days under the condition of 37xc2x0 C. and 5% CO2. Then, 3H-thymidine (3H-TdR) was added in a dose of 0.5 uCi/well. After 6 hours, the cells were harvested and subjected to the measurement of the uptake of 3H-TdR by a liquid scintillation counter. Thus, the DNA synthesis of pancreatic lymphocyte of mouse was measured was measured. The results are described as following table 1.
Experiment 2: Tumor metastasis inhibitory activity against B16 mouse melanoma.
B16 mouse melanoma cells (5xc3x97105 cells) were injected into tail vein of C57BL/6 mouse (7 weeks old). Next day, a solution which was prepared with several concentrations of the compound were injected into tail vein. After 14 days from the injection of B16 mouse melanoma cells, the mouse were killed under anesthesia and the lungs were excised. Numbers of nodules on the lung surface were measured and tumor metastasis inhibitory effects were estimated by comparison with control groups which were not injected the compound. The results are described as following table 2.